JMJD6 Shapes a Pro-tumor Microenvironment via ANXA1-Dependent Macrophage Polarization in Breast Cancer

Abstract

Breast cancer is the most common type of cancer in women worldwide, with the luminal subtype being the most widespread. Although characterized by better prognosis compared with other subtypes, luminal breast cancer is still considered a threatening disease due to therapy resistance, which occurs via both cell- and non-cell-autonomous mechanisms. Jumonji domain-containing 6, arginine demethylase and lysine hydroxylase (JMJD6) is endowed with a negative prognostic value in luminal breast cancer and, via its epigenetic activity, it is known to regulate many intrinsic cancer cell pathways. So far, the effect of JMJD6 in molding the surrounding microenvironment has not been explored.Here, we describe a novel function of JMJD6 showing that its genetic inhibition in breast cancer cells suppresses lipid droplet formation and ANXA1 expression, via estrogen receptor alpha and PPARα modulation. Reduction of intracellular ANXA1 results in decreased release in the tumor microenvironment (TME), ultimately preventing M2-type macrophage polarization and tumor aggressiveness.

Implications: Our findings identify JMJD6 as a determinant of breast cancer aggressiveness and provide the rationale for the development of inhibitory molecules to reduce disease progression also through the remodeling of TME composition.

Figure 1.

JMJD6 regulates ERα activity and affects prognosis in luminal patients with breast cancer. A, Kaplan–Meier analysis of patients with breast cancer included in the METABRIC dataset (available on cBioportal) showing the OS of all (left, n = 1,884), ERα-positive (middle, n = 1,440) or ERα-negative patients (right, N = 444). ‘High’ (red) and ‘low’ (blue) JMJD6 expression is shown according to median. P value was calculated with Log-rank (Mantel-Cox) test. B, Western blot showing the expression of JMJD6 and ERα in control (SCR) and JMJD6 KO MCF7 and MDA-MB231 clones. Vinculin is shown as loading control. C, Immunofluorescence staining of ERα (red) in scramble or JMJD6 KO MCF7 cells stimulated or not with E2. Nuclei were stained with DAPI (blue), bars = 50 μmol/L. D, Real Time PCR to evaluate the levels of ESR1 and (E) its target genes (PGR, BCL2, XBP1 and RARA) in control (SCR) of JMJD6 KO MCF7 stimulated or not with E2. Statistical significance was calculated by paired two-tailed t test comparing four independent experiments.

Figure 2.

Transcriptome profile of scramble and JMJD6 KO MCF7 and MDA-MB231 cells. A, Volcano plot showing statistical significance values against fold-change and highlighting the most upregulated (red) and downregulated (green) genes between JMJD6 KO and scramble MCF7 (top), and MDA-MB231 (bottom) cells. B, GSEA analysis of the most significantly up- or downregulated hallmark pathways in JMJD6 KO MCF7 (left) and MDA-MB231 (right) cells compared with scramble control. C, GSEA plots showing enrichment of genes associated with inflammatory pathways in JMJD6 KO MCF7 cells compared with scramble control. Significant GSEA pathways are defined on the basis of FDR < 0.05. D, mRNA expression of ANXA1 in WT, scramble and JMJD6 KO MCF7 and MDA-MB231 cells. t test was performed between JMJD6 KO MCF7 and scramble cells, and between JMJD6 KO and scramble cells. **, P < 0.01; ***, P < 0.001. E, Real Time PCR showing the levels of ANXA1 in control and JMJD6 KO clones. Statistical significance was calculated by paired two-tailed t test. F, Immunofluorescence staining showing the expression of ANXA1 (red) in scramble or JMJD6 KO MCF7 and MDA-MB231 cell clones. Nuclei were stained with DAPI (blue), bars = 50 μmol/L.

Figure 3.

ANXA1 is accumulated in LDs. A, IHC staining of ANXA1 combined with Oil Red O staining of WT MCF7 cells. Black arrows show colocalization areas. B, Oil Red O stained scramble or JMJD6 KO MCF7 and MDA-MB231 cells. Black arrows show LDs. C, Quantification of LDs shown in panel B. P values were calculated by unpaired two-tailed t test.

Figure 4.

PPARα regulates LD droplet formation and ANXA1 levels via ERα signaling. A, Real time PCR showing the expression of PPARα in control and JMJD6 KO MCF7 clones (left) and in MCF7 silenced for ERα (right). Graphs show the average of six independent experiments. P values were calculated by paired two-tailed t test. B, Western blot showing the levels of ANXA1 in MCF7 control or JMJD6 KO clones. Actin-β was used as loading control. C, ANXA1 ELISA performed with CM from control (n = 6) and JMJD6 KD (n = 3) and KO (n = 3) collected in independent experiments. P values were calculated by paired two-tailed t test. D, Fluorescence images showing the presence of LDs (HCS LipidTOX RED) in MCF7 and MDA-MB231 cells silenced for PPARα. Nuclei were stained with DAPI (blue), bars = 20 μmol/L. Graphs show the percentage of LD-positive cells/field; cells from at least six independent fields were counted. Statistical significance was evaluated by ordinary one-way Anova, multiple comparisons test. E, Fluorescence images showing the presence of LDs and ANXA1 (red) in scramble and JMJD6 KO MCF7 cells silenced for ERα. Nuclei were stained with DAPI (blue), bars = 20 μmol/L. Graph shows the percentage of LD-positive cells/field; cells from at least four independent fields were counted. Statistical significance was evaluated by ordinary one-way Anova, multiple comparisons test.

Figure 5.

ANXA1 released by tumor cells affects macrophage differentiation in vitro. A, Western blot showing the expression of JMJD6 in PyMT-41C cells in different KO clones. Actin-β was used as housekeeping protein expression. B, Volcano plot of statistical significance against fold-change between RAW264.7 macrophages cultured in CM of Jmjd6 KO or scramble PyMT-41C cells showing the significantly upregulated (32, red) and downregulated (1, green) genes. C, Heatmap shows the degree of clustering between biological replicates and distinct pattern of gene expression level between RAW264.7 macrophages cultured in CM of Jmjd6 KO (yellow) and scramble PyMT-41C cells (light green) cells. Color coding green to red shows the z-score value from -1.5 to 1. D, Relative fluorescence intensity (RFI) plots showing the expression level of M1 and M2 markers in M0, M1, and M2 BMDMs in CM of scramble and Jmjd6 KO PyMT-41C cells. Data were normalized on macrophages cultured without CM (red bar). Datapoints show the biological replicates of three independent experiments (green, blue and red) with two biological replicates in each experiment. Error bars show the SEM. P value is calculated with one-way ANOVA, Tukey multiple comparison test. E, Real-time PCR showing the expression of M1 markers (Cd40 and Nos2) and M2 markers (Cd206 and Arg1) in M1 and M2 differentiated BMDMs cultured in CM of scramble and Jmjd6 KO PyMT-41C cells, with or without the ANXA1 mimetic peptide Ac2–26.

Figure 6.

Tumor cell JMJD6 supports M2 macrophage differentiation and tumor growth in breast cancer mouse model. A, Tumor growth after inoculation of scramble (light gray) and Jmjd6 KO (dark gray) PyMT-41C cells in C57BL/6 mice. P value is calculated with two-way ANOVA, Sidak multiple comparison test. Error bars shows S.D. B, Tumor volume of scramble (light gray) and Jmjd6 KO (dark gray) PyMT-41C cells in C57BL/6 mice at the experimental endpoint of 35 days. P value is calculated with two-way ANOVA, Sidak multiple comparison test. Each dot represents a different mouse. Error bars shows SD among samples. C, Top, Representative images of two scramble PyMT-41C cells-derived tumors (clone A1 and clone B1) and two Jmjd6-KO PyMT-41C cells-derived tumors (clone A2A and clone C1A). Below, H&E staining of the different tumors. D, Percentage of CD45+ immune cells, CD11b+ myeloid cells, F4/80+ monocytes, macrophages, CD206+ M2-like macrophages over F4/80+ macrophages and PD-L1+ immunosuppressive macrophages over F4/80+ macrophages in tumors derived from scramble or Jmjd6 KO PyMT-41C cells. P value is calculated with unpaired t test. Each dot represents a different mouse. Error bars show SD among samples. E, IHC analysis of ANXA1 expression in tumors derived from scramble and Jmjd6 KO PyMT-41C cells injected in C57BL/6 mice.

Figure 7.

JMJD6 molds TME through ANXA1-dependent macrophages polarization. JMJD6 favors ANXA1 expression, intracellular accumulation, through a pathway involving PPARα and ERα, and its increase in the TME. ANXA1 released by cancer cells triggers the conversion of macrophages to a pro-tumor M2-like state which favors tumor development.